Recommendations on Organizational Changes, Technology Development, and Systems Approaches for Minimizing the Impacts of Huanglongbing and Other Diseases and Pests in Florida Citrus
Given the damage that citrus greening or huanglongbing (HLB) already has inflicted on the Florida citrus industry and the threat that HLB presents to the industry’s future, along with possibilities for invasions by new pests and diseases and for other challenges, a broad range of actions is recommended. The most critical needs are for immediate protection of citrus orchards against the advance of HLB, and our recommendations speak to this need. We also list other changes, which may be initiated in the near-term but are longer term in their execution, because they should result in improved disease control and crop productivity in the future.
In the context of this report, a near-term project is defined as having the potential to generate proof-of-principle or to demonstrate HLB mitigation in a period of 2 years or less. Intermediate-term and long-term projects are those with the potential to provide significant results in a time frame of 2–5 years and of more than 5 years, respectively. Our recommendations fall into the following four categories:
Organizational changes, designated O-1 through O-5 (Table 4-1)
Informational initiatives, designated In-1 through In-3 (Table 4-2)
Research and technology projects with near-term and near-to-intermediate-term potential, designated NI-1 through NI-11 (Table 4-3)
Research and technology projects with intermediate- and long-term potential, designated L-1 through L-4 (Table 4-4)
Recommendations within each of the above categories are presented in an approximate priority order. In making these recommendations, we are aware that some of them may correspond to actions that have already been initiated. We retain those recommendations for the sake of completeness, balance, and to indicate our support for such changes.
In the sections of the chapter that follow, the recommendations are described individually, by category, but because many recommendations are interrelated (within and across categories), some are discussed in relation to others and are cross-referenced in the text. Some activities are broadly enabling, and success in one area of research or effort can support the effectiveness of another activity. This reflects the nature of the overall strategy to try to achieve progress on multiple fronts and take advantage of synergies when possible.
RECOMMENDATIONS FOR ORGANIZATIONAL CHANGES
The concepts and technology of modern biology and related sciences will almost certainly be able to deliver sustainable methods for HLB mitigation not available and perhaps not even conceived at present. However, citrus production and quality must be maintained until new technologies can be put in place, or there will be no economic base to support implementing those technologies. Achieving an organized response to HLB will be a key to success in addressing the current, urgent needs of the citrus industry and developing the technology to sustain the industry in the future. Accordingly, recommendations for organizational changes are discussed first. However, the success of organizational changes is interdependent with informational initiatives and key research activities.
Recommendations O-1, In-1, In-2 and NI-1 have a high priority because of their potential for sustaining production until the to-be-developed approaches can be implemented. The committee recommends the creation of “Citrus Health Management Areas” (Recommendation O-1) to facilitate and coordinate control of ACP and the removal of affected trees, both in commercial orchards (Recommendation In-1) and in residential areas (Recommendation In-2). Recommendation NI-1 is intended to empower the Citrus Health Management Areas. This recommendation advocates for improved insecticide-based ACP control efforts, integrated with cultural practices, which are expected to improve management and minimize the effects on the environment and human health. Success under Recommendations NI-2 and NI-3, both concerned with achieving the detection of infected but asymptomatic trees, also has the potential to greatly improve the effectiveness of the efforts described in Recommendation O-1.
TABLE 4-1 Recommended Changes in Organizations Connected to Citrus Production
Create “Citrus Health Management Areas” in Florida.
Identify one organization and empower it to have oversight responsibility over HLB research and development efforts.
Create a centralized HLB website and data bank that is accessible to researchers and the public.
Commission an analysis of the economics of the citrus industry’s responses to HLB.
Organize an enhanced annual international symposium on all aspects of HLB.
Recommendation O-1 Create “Citrus Health Management Areas” in Florida to facilitate mitigation of HLB and other threats to citrus production.
Citrus production should be preserved against the immediate threat of HLB if efforts to achieve long-term and sustainable management are to have any value. We recommend the creation of Citrus Health Management Areas to coordinate management efforts in the near term. To operate effectively, a Citrus Health Management Area should have a relatively uniform incidence of HLB, i.e. disease pressure should be relatively constant across the area. The size should be 10,000–50,000 acres. A larger area favors more effective HLB mitigation but may
also require more intensive management if more growers are involved. Such areas would allow a small set of management strategies to be performed in an effective and timely fashion and their results to be evaluated. In addition, management strategies can be evaluated and compared among Citrus Health Management Areas with similar or different HLB incidences. Citrus Health Management Areas would be organized and operated by local growers and grower organizations with advice from an oversight organization (see Recommendation O-2). Funding for mitigation practices (i.e. insecticide applications and infected tree identification and removal) could come through grower taxes or possibly from government sources and be administered by the Florida Citrus Production Research Advisory Council (FCPRAC), the Florida Department of Citrus (FDOC) or the Citrus Research and Development Foundation (CRDF). The management areas would be charged with facilitating HLB mitigation and dealing with other threats to citrus production and quality within the area. The manager of a Citrus Health Management Areas should be empowered to enforce best management practices within the area, including cleaning-up abandoned or poorly maintained HLB-infected orchards, designing and implementing compensation plans, coordinating area-wide psyllid sprays, and reducing risk from infected urban citrus.
In some areas where incidence is low, aggressive tree removal and psyllid control will be the practice of choice. However, in areas where incidence is very high and tree removal is not feasible, only badly declining trees or blocks would be removed and those blocks with enough healthy trees to be profitable would be maintained for a few years. Such blocks would still need aggressive psyllid control to minimize spread to healthy trees or to less affected areas. In other areas, it may be necessary to remove as much citrus as possible so that new plantings can be made without the danger of being surrounded by abundant sources of inoculum. Removal of badly affected groves can result in loss of the agricultural tax exemption. Groves would need to be converted to pasture or planted with forest trees to maintain that exemption. Changes in tax law could be helpful in eliminating this impediment to removal of affected groves.
Area-wide insect control strategies should utilize “window” strategies where only certain classes of insecticides are permitted during specific periods to minimize the development of resistance (see Recommendation NI-1). Programs designed to train, and incentives to retain, personnel should be developed to ensure continuity of personnel familiar with needs of that region.
Research has shown that low volume aerial applications are the most convenient way to achieve Asian citrus psyllid (ACP) control in the thousands of acres of a Citrus Health Management Area. However, while aerial application for other arthropods requiring management within the area (e.g. mites) may be combined with ACP insecticides, possible effects on non-target natural enemies and other pest species must be taken into account. Care and knowledge are needed to determine which insecticides can be effectively used in low volume aerial applicators.
Control of ACP in organic orchards is challenging since there are no systemic products approved by the Organic Materials Review Institute (OMRI). This leaves products such as JMS Stylet oils and products based on neem and chrysanthemum as the only choices, and these are not likely to result in the effective long-lasting control needed to mitigate HLB. Any failure to control ACP and HLB in an orchard has direct negative impacts on surrounding orchards.
Recommendation O-2. Identify one organization, preferably an existing organization, and empower that organization to have oversight responsibility for research and development efforts intended to improve management of HLB and to plan for the future of citrus production in Florida.
It is not possible (because of diverse funding sources), and likely not desirable, to bring every funded HLB research project under the same roof. A competitive grants program should be maintained as the primary tool for enabling individual investigators to take up new approaches to HLB mitigation. However, greater coordination of projects and project funding will be beneficial, particularly as projects advance to the phase of field testing and intellectual property issues arise. Among the objectives of the proposed empowered organization would be the following: (a) Identify pre-competitive and non-competitive research goals and manage industry-sponsored research to advance those goals; this would include terminating specific projects before their ending date if appropriate; encouraging and coordinating collaborative research efforts related to these research goals at the national and international levels, including incorporating Citrus Health Response Program (CHRP) efforts into research planning; (b) Oversee the testing and comparison of new disease mitigation technologies (see Recommendations NI-2 and NI-3) and provide advice in the development of Citrus Health Management Areas; (c) Manage regulatory and intellectual property, and submissions to and negotiations with regulatory agencies, related to citrus cultivars and disease control technology; (d) Provide for public education on citrus issues; (e) Assist in guiding lobbying objectives of Florida Citrus Mutual and other citrus grower organizations, and (f) Develop 5-year, 10-year, and 15-year plans for the future advancement of the industry.
The science management responsibilities of the proposed organization require expert input with a minimum of bias. It is critical that a scientific advisory board (SAB) be appointed. The majority of the SAB membership should consist of scientists who are not active citrus researchers. Among the responsibilities of the SAB should be to recommend research priorities, to review and prioritize research grant proposals, participate in an annual HLB international symposium, and to analyze research progress biyearly, including consideration of requests from PIs to change the direction or emphasis of the project. Through its review of research proposals and progress reports and its participation in the annual research symposium, the SAB will be in an ideal position to notice new developments that could contribute to synergistic interactions between research projects. The SAB should be charged with being alert to such possibilities.The SAB would make recommendations for continuation or re-direction of the project, or, in those rare instances when there seems to be little or no hope of further benefit, for termination of the project.
Recommendation O-3. Create a centralized HLB website and data bank with geographical HLB-incidence and other data, accessible to researchers and the public.
HLB is now present in several southern-tier U.S. states, Mexico, and Caribbean countries; other U.S. states have reported ACP findings, although not the HLB-associated pathogen, Candidatus Liberibacter asiaticus (CLas). The proposed website should include a database of the occurrence and distribution of the ACP and HLB in the United States and a bulletin board, which would serve as a source for information on scouting and sampling techniques, management recommendations, pesticide application technologies, summaries of recent research results, and other relevant content. The website should be accessible to researchers and the public and be
linked to the CHRP and the University of Florida’s Institute of Food and Agricultural Sciences (UF-IFAS) websites. The website could be modelled after other sites with similar audiences, such as the US Department of Agriculture Integrated Pest Management (IPM) Soybean Rust PIPE (Pest Information Platform for Extension and Education) at http://sbr.ipmpipe.org.
Recommendation O-4. Commission an analysis of the economics of the citrus industry’s responses to HLB, including cost-benefit analysis and the potential of new technological developments.
Examples of the types of analyses that will be of value are: (a) cost-benefit analysis for different areas of Florida for current practices of HLB orchard inspection and tree removal; (b) anticipated research costs and potential impacts of new technological approaches to HLB mitigation and new methods for orchard inspection; and (c) relative cost analysis for alternative policies and orchard practices intended to minimize the impact of declining citrus quality and declining, or even collapsed, citrus production due to endemic HLB. The field of health care economics has developed sophisticated, but practical and both prospective and comparative, technology assessment models and simulations. These analytical tools could complement currently used agricultural methods and, in concert, may help guide HLB management strategies and investments.
Recommendation O-5. Organize an enhanced annual international symposium on all aspects of HLB.
There are about one hundred HLB-related active research projects in the United States. Methods, results, and concepts developed in one project will in many instances be unknown to researchers in other projects whose efforts could benefit if they did know of them. In many instances, collaborations between groups could have synergistic effects. Information transfer and the realization of the possibilities for collaboration are two of the obvious benefits of bringing researchers from different HLB projects together at an annual international research symposium. Experience shows that sometimes such contacts result in ideas for new and productive research directions not obvious to members of the separate projects. However, the benefits of gathering project researchers together can be leveraged and enhanced by also inviting prominent experts, particularly those whose accomplishments include the translation of research results into products and services. These experts, typically in fields directly or tangentially related to citrus research, would hear reports, interact with project researchers, and provide comments and suggestions. The SAB (described in Recommendation O-2) should be requested to prepare a report of the main advances presented at the symposium and actions that should be considered because of reported results, with the goal of accelerating the translation of research results into HLB-mitigation practices. This meeting should also be attended by the heads of the major funding agencies from Florida, Brazil, and other citrus-producing countries with major research programs on HLB. These representatives should meet and closely examine the projects funded by each to enable coordination of research efforts, avoid duplication, and enhance cooperation among investigators.
INFORMATIONAL INITIATIVES: COMMUNICATING TO THE PUBLIC AND PUBLIC OFFICIALS THE SERIOUSNESS OF HUANGLONGBING AND OTHER THREATS TO FLORIDA CITRUS PRODUCTION AND THE COUNTERMEASURES THAT MAY BE NEEDED
Most public officials who are involved in agriculture or the funding of agricultural research projects are well aware of, and reasonably knowledgeable about, the status of HLB and its implications for the citrus industry and the broader economy of Florida. However, there are individuals and groups whose actions and decisions could have an impact on HLB management but who do not have sufficient understanding of the potential long-term effects that the HLB problem could have on the economy. The need for prompt detection and removal of infected trees, and the rigor required to mount an effective HLB management program, are still not appreciated by many growers. Some homeowners are aware of the HLB problem, but do not recognize that HLB-affected trees in their landscape plantings may be contributing to the situation and that the removal of those trees could be helpful. Officials, especially at the county and city level, are often not aware of the seriousness of the problem and its implications for the economy of their area. Table 4-2 lists initiatives for communicating information about HLB to different audiences.
TABLE 4-2 Recommended Informational Initiatives
Expand extension efforts emphasizing the importance to HLB management of removing infected trees from groves.
Encourage homeowners to remove and properly dispose of backyard citrus trees, particularly HLB-affected trees.
Communicate information on HLB and its potential economic impact to government officials at the federal, state, county, and city level.
Recommendation In-1. Expand extension efforts emphasizing the importance to HLB management of removing infected trees from groves.
To the extent possible, recommendations for HLB mitigation, through inoculum removal and ACP management, should be agreed upon among researchers and extension agents and implemented uniformly in the Citrus Health Management Areas (Recommendation O-1). Current differences of opinion among extension agents have been detrimental to HLB management, causing confusion among growers. Programs for management of psyllids are advancing and improving rapidly, but there is a general lack of appreciation of the importance of rapid detection and removal of affected trees for mitigation of HLB. The extended latent period between infection and symptom development in citrus means that incidence may continue to rise for 2 to 3 years after a program is initiated, even if the program followed is rigorous. The early rise in incidence gives the incorrect impression that removal of infected trees is ineffective and makes it difficult to justify the several hundred dollars per acre annual cost of continuing the program.
In addition, the application of micronutrients and materials that induce systemic resistance has been promoted by some growers and extension personnel. However, there is no firm evidence that these measures do more than temporarily improve tree appearance, and it is likely that they are counter-productive because they leave sources of inoculum, in the form of
symptomatic trees, in place in the grove. The use of micronutrients and systemic resistance agents should be discouraged by extension agents unless substantial evidence of the efficacy of these agents is derived from research projects with appropriate controls. Training programs by the Extension Service for HLB scouts in the detection of HLB and scouting methods would be useful in the effort to reduce CLas inoculum.
Recommendation In-2. Encourage homeowners to remove backyard citrus trees, particularly HLB-affected trees.
CLas-infected citrus in residential areas contributes significantly to the inoculum available for spread of the disease in commercial citrus. Homeowners, in general, are unaware of HLB, that their infected tree soon will be useless, or the importance to the citrus industry of removing infected trees promptly. In general, the suburban setting is not well suited to the implementation of the intensive disease control practices needed for long-term citrus culture, and homeowners should be encouraged to replace citrus with other species to reduce potential reservoirs of disease. The Extension Service should use the news media and Internet to help inform homeowners about HLB, encourage removal of affected citrus and related ornamental species such as Murraya, and provide information on citrus substitutes to help discourage planting of citrus in the home landscape. Information on the availability of citrus substitutes should be publicized by the Extension Service. At some point in the future, resistant varieties of citrus may be available and allow re-establishment of citrus in the home landscape.
Recommendation In-3. Communicate information on HLB and its potential economic impact to government officials at the federal, state, county, and city level.
Citrus industry leaders and the Extension Service need to do more to inform local officials about the threat posed by HLB. The citrus industry has faced serious challenges in the past that have been publicized, but the industry has always been able to deal with them. In this case, even under the best scenario, HLB is likely to change the industry permanently. Public officials will have to deal with the consequences of those impacts in terms of the local economy, employment possibilities, and reduced tax receipts.
RECOMMENDATIONS FOR RESEARCH AND TECHNOLOGY WITH THE POTENTIAL TO ADVANCE HUANGLONGBING MITIGATION IN THE NEAR-TERM OR NEAR-TO-INTERMEDIATE-TERM
The near-term efforts in HLB mitigation are focused on (a) clean-stock programs; (b) ACP suppression; and (c) reducing sources of inoculum. The use of protected environments, such as screen houses, for citrus budwood production is now accepted practice in the industry and legally mandated, so we do not comment on clean stock programs here. Table 4-3 lists recommendations for research and development that are likely to generate significant results in 2 to 5 years. Some of these, particularly NI-1 (and its subcomponents), NI-2, and NI-3 will be important to implement in synchrony with organizational changes and informational initiatives. They are aimed at improving the effectiveness of ACP suppression and inoculum reduction in the short-term. Recommendation NI-7 is also focused on ACP suppression, but will be scientifically challenging to achieve. Recommendations NI-4, NI-5, NI-6, and NI-8 through NI-11 are directed at making the investments that may generate useful novel concepts for HLB mitigation.
TABLE 4-3 Near- and Near-to-Intermediate-Term Recommendations
Improve insecticide-based management of ACP.
Support searches for biomarkers that may be exploited to detect CLas-infected citrus.
Establish citrus orchard test plots for evaluation of new scouting and therapeutic methods.
Accelerate the sequencing, assembly, annotation and exploitation of a sweet orange genome to provide a powerful tool for all future citrus improvement research.
Support development of HLB model systems.
Exploit the CLas genome sequence for new strategies of HLB mitigation.
Support research aimed at developing alternative ACP management strategies.
Support small-scale studies on the feasibility of alternative horticultural systems suited to endemic HLB.
Support demonstration of RNA interference (RNAi) effects for possible suppression of ACP.
Develop in vitro culture techniques for CLas to facilitate experimental manipulation of the bacterium for insights into gene function.
Sequence, assemble and annotate the ACP genome to provide a basis for new approaches to ACP management.
Recommendation NI-1. Improve insecticide-based management of ACP.
Approaches recommended here, applied in concert, will support Recommendation O-1, which advocates creation of Citrus Health Management Areas in Florida to facilitate mitigation of HLB and other threats to citrus production. The suppression of ACP populations by insecticide application will be a necessary part of HLB mitigation for the foreseeable future; e.g., until sources of inoculum can be drastically reduced (Recommendations NI-2 and NI-3) or CLas-resistant citrus (see Recommendation L-1) becomes available for planting. We strongly support the idea that present management strategies can be improved by gaining better understanding of ACP and CLas, their ecology and epidemiology, and the presently available (or soon to be available) tools that can aid in their management. Recommendation NI-1 is concerned with improving conventional insecticide suppression of ACP by integrating four types of research and development activities:
Recommendation NI-1a. Develop new methods to fine-tune field surveillance of ACP via more efficient and consistently applied trapping or other methods, to improve timing and targeting of insecticide applications.
At the moment ACP populations are monitored differently in different regions. Additional effort should be invested in developing more effective traps that will provide an accurate assessment of the ACP population so sprays can be more effectively timed. Sampling methods also should be refined within and between areas to help describe actual population trends and help in area-wide management strategies. Monitoring and analyzing ACP populations over the
course of each year is also important for observing long-term trends and examing their relationship to environmental factors. Surveillance should not be limited to active orchards. Declining or abandoned orchards and citrus in urban landscapes should also be monitored to identify local ACP populations for treatment before they disperse. Well-timed applications and proper use of insecticides, based on surveillance for ACP and expected year round variations in ACP populations, present an opportunity to maximize insecticide effectiveness while minimizing risks.
Recommendation NI-1b. Evaluate new insecticides, adjuvants and application methods for their effectiveness against ACP or HLB infection.
It is critical for near- and intermediate-term mitigation of HLB to identify effective new insecticide active ingredients and adjuvants and to critically assess application techniques (soil, trunk, and aerial; low-volume, airblast, and other advanced methods) for both adult and nymphal stages of ACP. Of particular interest are insecticides with good safety profiles and with chemistries distinct from those of insecticides currently in use on citrus. Many new adjuvants have characteristics that enhance their penetration into the tree canopy and uptake in the leaves. Criteria for evaluation of application methods should include enhancement of insecticide effectiveness, ability to cover large areas quickly, compatibility with frequent HLB monitoring and low application costs. The citrus industry should foster greater collaboration and partnerships with the pesticide industry, and growers and researchers should continue to work with the federal minor-use pesticides program (also known as IR-4) and take advantage of legislation that will help evaluate the effectiveness of, and obtain needed registration data for, insecticides critical to the citrus industry. The need to conserve beneficial organisms, with due consideration to the economics of the production system, is already appreciated by pest control personnel but needs to be emphasized.
As ACP populations increase, it is likely that some insecticide resistant ACP strains will develop. There is anecdotal evidence that resistance to neonicotinoids may be starting to develop. This is troubling because neonicotinoids are being used both as a systemic drench for longer-term control on young trees and as foliar sprays. Insecticides, particularly those that have proven to cause only minimal harm to the environment and human health, must be protected from becoming obsolete because of resistance development, such as that caused by inappropriate season-long application of neonicotinoids or their use as foliar sprays. One of the advantages of the proposed Citrus Health Management Areas (Recommendation O-1) is that they can focus on minimizing development of insecticide-resistant ACP.
Recommendation NI-1c. Investigate the behavioral ecology and CLas transmission biology of ACP to improve timing and other aspects of insecticide application.
Research and practice in other systems suggests that increased understanding of insect behavioral ecology can facilitate insect management and mitigation of insect-transmitted pathogens. Research on aphids revealed the timing of dispersal flights, including mass migrations, and suggested strategies for successful monitoring of aphid populations. ACP is a new pest in Florida, and our insufficient knowledge about its interactions with its new environment limits the ability to manage ACP effectively. Near- and intermediate-term research efforts should be able to gather data on the interactions of ACP (both adults and immature
forms), with CLas, plants, and the environment that can improve the effectiveness of insecticide usage and reveal points in the ACP life cycle that may be vulnerable to interdiction.
No definitive study has been published regarding ACP flight activity, e.g., flight range and seasonality, which would provide the ability to predict times at which adults are likely to migrate and generate information for epidemiological models. Convenient measures of macro-scale aspects of ACP behavior, such as the attractiveness of intact orchard trees for ACP are not available. Results from these studies would provide a rational basis for seeking and identifying behavior-modifying natural and synthetic products for managing ACP. At the very least, study of ACP flight behavior, sex attraction (Wenninger et al., 2008), or orientation to its host plant (Wenninger et al., 2009b) should yield data useful for improving methods to monitor psyllid movement.
Insect developmental processes and population increase are temperature driven in major part, so knowledge of local temperatures is a critical variable. Developmental and nutritional status of the host plants and climatic conditions (e.g., humidity and rainfall) are also very important. For example, flushes of new growth in citrus are important for influencing ACP flights and reproduction success. Other environmental factors may influence the timing and efficiency of long distance versus short distance spread, which can inform decisions on establishing distances between HLB-affected old orchards and replantings.
CLas infections may influence ACP behavior. For example, the increased carbohydrate accumulation characteristic of CLas-infected plant tissue may be a CLas adaptation mechanism that favors ACP feeding and oviposition on infected tissue and therefore enhanced acquisition of CLas. The circulative/propagative transmission mechanism likely to be involved in ACP-CLas interactions requires delivery of CLas into the host in saliva. Understanding salivation physiology or behavior might reveal targets for plant genetic engineering or other approaches to preventing ACP feeding.
Compared to the adults, ACP nymphs acquire CLas efficiently and they may be more susceptible to management efforts. Unlike adults, nymphs excrete honeydew as waxy-appearing filaments. This form of excretion most likely evolved to protect tightly clustered immatures from fouling one another with sticky honeydew. The effect of these secretions on natural enemies should be investigated. Since the waxy excretion is a waste product from ACP digestion and metabolism, analysis of this material can be used as a bioassay for compounds that have negative affects on nymph development. Detailed observations of immatures as they develop from egg stage to adult should be conducted in the field and greenhouse (e.g., by video recording). Important characteristics are the spatial dynamics of nymphs over time, excretory behavior, interactions with parasites, predators and other biotic stress, interactions, if any, with ants, and effects of rain, wind and other weather-related factors on aggregations. This information should be acquired for both a highly susceptible host (e.g., sweet orange) and a poor or marginal host (e.g., mandarin orange). Depending on results from the above, evidence of pheromones (alarm, marking), acoustic signals or other stimuli may be obtained.
Additional knowledge is needed about the ability of ACP to acquire and inoculate CLas. For example, how efficient can asymptomatic infected trees be for CLas acquisition by ACP? Orchard test plots including such trees (Recommendation NI-3) could be an experimental tool for answering this question. Some reports suggest that the latent period between CLas acquisition and inoculation by ACP may be as short as 8 days or as long as 25 days. Is the time between ACP acquisition of CLas and becoming inoculative really highly variable, or can it be predicted based on environmental conditions? The spread of HLB may be greatly reduced if insecticides
can be applied to the ACP population prior to it becoming inoculative and prior to its movement to a new host.
Although the existence of unrecognized reservoirs of CLas inoculum has been postulated, the spread and accumulation of HLB would seem to be accounted for by infected ACP and citrus species. Therefore, requests for support of research on other reservoirs should be considered only if they are based on preliminary evidence that such reservoirs could be of epidemiological significance.
As more information on ACP behavioral ecology and CLas acquisition and retention in the vector is accumulated, incorporation of the data into a mathematical model may allow prediction of ACP movement patterns. This will be a longer term effort (see Recommendation L-3).
Recommendation NI-1d. Evaluate new cultural practices for their effectiveness against ACP or new HLB infections.
A list of practices that can be used to reduce ACP and HLB is included in the 2010 Citrus Pest Management Guide (Rogers et al., 2010). These include practices that reduce the occurrence of new growth flushes and treatment of infected trees with a quick-acting insecticide to reduce the potential for ACP to move to other hosts before the trees are removed. These practices should be augmented with new practices where appropriate, be implemented over an entire Citrus Management Health Area, and be evaluated for effectiveness.
Discussion of the Merits of NI-1
Favoring rationale: In Florida, there is considerable existing expertise on insect management, the management of other pests, insect ecology, and the modification of cultural practices, such as management of flush. There are many insecticides registered and recommended for ACP in Florida (Rogers et al., 2010), and this situation is favorable for development of effective IPM programs for ACP in Florida citrus. Florida entomologists are generally prepared to collect the types of information described in Recommendation NI-1c on adult and immature forms of ACP and the interactions of ACP, CLas, citrus trees and the environment. The ACP immature stages are especially under studied. Since late stage ACP nymphs are far more efficient at acquiring the HLB bacterium than adults, it is logical to look closely at their development, ecology and behavior with an eye towards interfering with nymph development. Since the immatures may be more readily manipulated than adults, they could be more vulnerable to management practices. Better integration of research and application efforts can produce a whole that is greater than the aggregate of its parts.
Disfavoring rationale: It is not apparent that there is a sufficient level of cooperative effort between research groups to achieve the goals of this recommendation. Additionally, the occurrence of citrus trees whose owners choose not to apply effective insecticides is a significant impediment to effective ACP management. Examples are abandoned and semi-abandoned groves and backyard ornamental citrus as well as citrus in organic production. Any failure to control ACP in a given setting will likely have direct negative impacts on surrounding commercial orchards. Vigorous ACP suppression programs may include applications of aldicarb, a very effective insecticide but one that is of concern to human health and to the environment.
Examples of currently or recently supported research projects and relevant results: There are several currently funded projects that address insecticidal practices for ACP. These include at least two projects investigating ultra-low volume techniques for applying foliar insecticides over
a wide area quickly, three projects investigating different rates and different insecticides for seasonal control of ACP, one project investigating the use of citrus flushes and dormant spray oils for ACP control, and another investigating cross-resistance between insecticides and the potential for evolution of resistance.
Examples of possible future research projects: It is likely that significant benefits to management of ACP will arise from technologies that: (a) help time insecticide treatments through the use of ACP monitoring tools; (b) apply the insecticides quickly over large areas and utilize techniques that ensure effective coverage; (c) deliver insecticides that provide maximum effectiveness against ACP with maximum safety to applicators, consumers and the environment; (d) minimize the development of resistance to insecticides; (e) conserve beneficial organisms within the citrus agroecosystem; and (f) are adaptable to application under Recommendation O-1 to the Citrus Health Management Areas. Registration of insecticides that are effective when applied with low volume applications will be crucial to ACP management since this will allow large areas to be treated rapidly when needed. Alternative insecticides with equivalent efficacy to aldicarb are urgently needed. It is important that results from research on insecticide management of ACP be conveyed through extension services and private consultants. This would be best achieved by involving them directly in the research trials so they are full partners.
Suggested support mechanism: Projects in support of alternative management strategies should be funded by both Florida programs and USDA programs such as Pest Management Alternatives Program (PMAP) and IR-4. It is reasonable to expect insecticide manufacturers to also provide support for such projects.
Time to outcome: Based on the experience of the Brazilian citrus industry and in one area of Florida, investments in improved insecticide applications have great potential for near-term, as well as intermediate-term, benefits to ACP management.
Other sections of this report containing related information: Chapter 2, pp. 34–37 (transmission by ACP); pp. 47–49, (ACP biocontrol and guava as ACP repellent); pp. 53–55 (insecticidal control); Chapter 3, pp. 72–77 (reducing ACP access to citrus); Appendix J; Appendix K (insecticide and spray effects on ACP mortality).
Recommendation NI-2. Support searches for biomarkers that may be exploited to more efficiently detect CLas-infected citrus, with emphasis on identification of asymptomatic infected trees.
Visual surveillance by trained personnel is now the only reliable and cost effective method for identifying potential reservoirs for HLB, both in and out of the orchard. Trained scouts can detect symptoms of HLB-infected trees with reliability, but typically not until 6–18 months or possibly years after the infection of citrus with CLas by ACP and the tree has become a potential reservoir for spread of HLB. Automation of the detection of symptomatic infections would be of great value (e.g., in allowing increased frequency of scouting at a lower cost). Compared to a symptomatic tree, a CLas-infected asymptomatic tree on average is (a) available as a CLas source for fewer months; and (b) has a lower CLas titer and proportion of the tree invaded by CLas. Therefore, the ability to detect CLas infections at reasonable cost in asymptomatic trees could have a revolutionary effect on HLB mitigation practice and effectiveness, especially, but not only, in areas of low HLB incidence with large plantings of citrus that are away from urban areas or poorly maintained/abandoned groves. ACP suppression efforts might be relaxed to some extent if CLas reservoirs could be more effectively eliminated.
Identifying CLas-infected orchard citrus trees may rely on detecting CLas itself or on observing the signal of an infection-related altered-state signal of the tree, a biomarker, that would telegraph the presence of infecting CLas. Highly sensitive and specific means for detecting CLas are available. However, CLas is unevenly, highly locally, and unpredictably distributed in an asymptomatic tree (Tatineni et al., 2008), creating an unmanageable sampling problem for detection. Therefore, finding asymptomatic infected trees by CLas detection will require new information on the patterns of infection within trees. Tree cytological and physiological changes (Etxeberria et al., 2009; Folimonova et al., 2009) not suitable for use as biomarkers directly nevertheless may enhance our understanding of CLas spread in the tree, guiding sampling for CLas detection and the development of new biomarkers.
The spatial and temporal aspects of CLas invasion of the tree and their relationship to the spatial and temporal development of biomarkers, especially prior to appearance of symptoms, will be an important basic component of research advancing this recommendation. How is the movement of CLas in the tree related to symptom development and the activation of relevant biomarkers? Are there cytological changes such as phloem necrosis which, though not useful as an easily assessed biomarker, can be used to trace the movement of CLas? Results from investigations related to these questions could enhance sampling procedures for more efficient detection of asymptomatic, infected trees.
CLas-infection specific biomarkers, should they be discovered, have the potential to be detected in parts of the tree not reached by the CLas infection, thus increasing the probability of detection. Three classes of potential CLas infection-revealing biomarkers are volatile organic compounds (VOCs) emitted by the tree, optical changes in the tree, and changes in the concentrations of small molecules or of proteins, nucleic acids or other macromolecules in the tree tissue. Only the rare biomarker provides a strong and unequivocal signal; typically the detection requires instruments of high sensitivity, high specificity, and ease in use.
VOC biomarkers: The potential of VOC measurements to detect plants entering a state of biotic or abiotic stress is widely recognized. VOC detection in the parts-per-trillion range probably will be required, a sensitivity that has been achieved by dogs and bees and, recently, by the relatively new differential mobility spectrometry (DMS) instruments (Krebs et al., 2005; Molina et al., 2008; Zhao et al., 2009). In addition to direct searches for VOC biomarkers, analysis of changes in gene expression as revealed by DNA arrays, or gene chips, can be interpreted using state-of-the-art bioinformatics procedures to discover infection-specific alterations in metabolic networks that are predictive of changes in proteins and small molecules, including tree volatiles.
Optical biomarkers: Reflectance measurements, and more generally the full range of biophotonics techniques, have the potential to distinguish uninfected from infected trees, even trees that appear not to be infected. Even an optical scanning method of limited capabilities could be of value; for example, a method that can detect infected trees with a significant rate of Type I (false positive) errors (e.g., due to non-HLB disease conditions), but that has a negligible rate of Type II (false negative) errors. Application of such a method would be followed by targeted visual inspection or PCR analysis rather than a full tree-by-tree survey.
Chemical biomarkers: In several systems, induction of plant innate immune response has been reported to occur early in the infection process, before the appearance of symptoms, and was detectable locally as well as at more distant points in the plant (Boller and Felix, 2009). Whether any pre-symptomatic biomarker spreads uniformly or to specific locations in citrus trees is not known but can be readily investigated. The number of trees, and the probable requirement
for multiple samples per tree, eliminate traditional wet chemical methods as viable candidates for biomarker detection. However, chemical sensing electrodes and chemically reactive “dip sticks” could eliminate or minimize extraction procedures.
One often proposed approach to CLas-infection detection is the planting and surveillance of “sentinel” plants. Compared to citrus, a sentinel plant would need to be more attractive to ACP and show symptoms of CLas infection sooner. CLas has been detected in individual ACP collected in orchards that were without visibly infected trees (Manjunath et al., 2008). Technology that will probably detect CLas-infected but asymptomatic orchard citrus trees or CLas-infected ACP will be more effective than a sentinel plant at revealing the presence of HLB, especially since a requisite sentinel plant species has not been identified.
Discussion of the Merits of NI-2
Favoring rationale: In-orchard detection of biomarkers as tree volatiles, by optical methods, or by non-traditional wet chemical methods has the potential to augment and revise current scouting practices and allow more efficient and frequent surveillance to achieve unprecedented reductions in CLas reservoirs. Recent improvements in the sensitivity, specificity, portability and ease of use of instruments suited to biomarker detection favor a look at new detection technologies at this time.
Disfavoring rationale: Although DMS and other sensitive instruments for detecting VOCs have been developed and some have been significantly miniaturized, none are suited at this time for direct in-orchard detection of trees with altered VOC emissions. Increases in the quantity of VOCs released by the biologically stressed plant are more common than the production of new VOCs, which would be more easily detected than quantitative changes. The only high-sensitivity, high-specificity method for detecting CLas infections in asymptomatic trees is by conventional PCR analysis, which currently would be impossible to apply on an orchard scale for the 50 million citrus trees in Florida. It does not appear that any optical measurement has been able to distinguish HLB from other citrus maladies. Differences in chlorophyll content appear to be responsible for the most prominent changes in spectral reflectance of leaves from HLB and healthy citrus. Changes in chlorophyll content may have a variety of causes not related to HLB. Although experiments with greenhouse-grown citrus trees suggest that biomarkers of asymptomatic CLas infection have been observed, orchard citrus trees are very large compared to experimental greenhouse specimens and are subject to a greater range of biotic and abiotic stress, so biomarkers discovered in greenhouse experiments may not be relevant to orchard conditions.
Example of currently or recently supported research projects and relevant results: Research projects in which gene expression, protein accumulation, or metabolite composition are compared for CLas-infected and non-infected citrus are in progress. All have the potential to reveal new CLas infection biomarkers. References cited above identify projects aimed at discovering VOC and optical biomarkers.
Examples of possible future research projects: Although not a research project, if the orchard test plots described in Recommendation NI-3 are created and made available to researchers, it will be possible to rapidly evaluate biomarkers of all three types described here as well as any instrumentation designed to exploit those biomarkers.
Suggested support mechanism: It is possible that the detectors and software needed for discovery of CLas-infected citrus trees using optical or VOC biomarkers already are available in a university, institute or government laboratory or at a commercial firm. The offering of an
inducement prize may activate the shortest path to deployment of CLas-infection-detecting equipment, with the Recommendation NI-3 orchard test plots serving as the testing ground for demonstrating successful detection. Competitive grants funding should be continued in support of biomarker discovery.
Time to outcome: Near- to mid-term for discovery of suitable biomarkers, mid-term for development and deployment of instrumentation for improved HLB scouting.
Other sections of this report containing related information: Chapter 3, pp. 69-71 (removing HLB-affected trees).
Recommendation NI-3. Establish citrus orchard test plots that include CLas-infected but asymptomatic trees, as well as symptomatic trees, for evaluation of new scouting methods and therapeutics.
The establishment of test plots is intended to provide a systematic approach for realistic evaluations of methods for early detection of CLas-infected trees, as described in Recommendation NI-2, and for evaluating the efficacy of potential therapeutic treatments. It is crucial that new scouting methods and new therapeutics, already developed to the point of showing promise and perhaps proof of concept under laboratory or greenhouse conditions, be examined under orchard conditions with suitable controls before further investments of time and money are made. As an example of the need for this information, a therapeutic treatment that improves the appearance of trees but fails to reduce CLas titers can have serious consequences because CLas reservoirs would be maintained rather than removed.
Test plots could be established and maintained under contract and with cooperation from growers or owners. A plot may, for example, be located in an isolated, abandoned grove. Each test plot should be in a different region or environment, so that the general effectiveness of therapeutic agents can be assessed and biomarkers specific for CLas infection can be validated to be context-independent. Each plot should have trees documented to be (a) infected and showing HLB symptoms; (b) most importantly, CLas-infected but asymptomatic; and (c) uninfected, to the extent that freedom from CLas can be verified.
Identifying infected but asymptomatic trees probably will require research effort and may involve extensive sample collection from suspect trees and sensitive and high-throughput PCR analysis. Alternatively, a surrogate for an asymptomatic tree might be prepared by pruning out symptomatic branches. Less realistic, but possibly useful for preliminary tests, would be artificially inoculated trees in the greenhouse or in the orchard. Investigators may be able to find other ways to apply current, and likely arduous, methods to identify a small number of infected but asymptomatic trees in existing citrus orchards.
Over time, asymptomatic trees will show symptoms, and uninfected trees will become CLas-infected. Therefore, assessments of infection state should be carried out periodically, and a database recording the infection state of trees in the test plots will need to be updated accordingly. Plots should be made available for use at cost to qualified and responsible investigators and competitors for inducement prizes (see Recommendation NI-2, Suggested Support Mechanism). The infection state of specific trees would be confidential information to be revealed only after scoring by investigators and competitors have been completed.
Discussion of the Merits of NI-3
Favoring rationale: It is critical and urgent to determine whether compounds and mixtures considered to have therapeutic activity and biomarkers intended to reveal early CLas infection are actually effective and to do so at a minimum of expense and with convincing outcomes. Only a systematic approach with centralized test sites can accomplish this.
Disfavoring rationale: Although the accumulation of CLas has been assessed in infected, symptomatic orchard citrus trees (Li et al., 2009; Trivedi et al., 2009), the distribution of CLas in trees at early stages of infection is undocumented. In the absence of such documentation, large numbers of candidate trees may be required in order to assure the presence in the sample of a sufficient number of infected but pre-symptomatic trees, significantly increasing the costs of creating and maintaining the reference plots. Whether information on, and statistical analyses of, the spread of HLB in groves will be sufficient to assist in the design of the reference orchard is unknown. The establishment of HLB reference plots will be antithetical to HLB mitigation practices in many areas, limiting the choices of available plots.
Example of currently or recently supported research projects and relevant results: None known.
Examples of possible future research projects: The HLB reference citrus orchard would be a service to researchers and technologists rather than a research project.
Related information and references: As indicated in Chapter 3, the average titer of CLas in infected but asymptomatic trees is lower than it is in symptomatic trees, and, under greenhouse conditions, the CLas titer may be more than 104 greater in a symptomatic tree than an asymptomatic tree at an early stage of infection. Where the titer is still very low, the tree should have a significantly diminished ability to serve as an HLB source compared to symptomatic trees.
Suggested support mechanism: Creation, assessment and maintenance of HLB reference citrus orchard plots should be under the control of a central authority, presumably under supervision of a contractor or a research organization. Detection of CLas-infected asymptomatic trees should be by certified methods and be subject to review by state or federal authority because the specification of “infected” could be subject to contention, especially where an inducement prize is at stake.
Time to outcome: Establishing HLB reference citrus orchard plots should be initiated as soon as possible because there is a critical need for surveillance technology capable of more rapidly and efficiently detecting symptomatic trees than current scouting practices allow, and facile detection of infected but asymptomatic trees would revolutionize HLB mitigation practices.
Other sections of this report containing related information: Chapter 2, pp. 49–50 (survey methods), and pp. 67–69 (removing HLB-affected trees).
Recommendation NI-4. Accelerate the sequencing, assembly, annotation and exploitation of the sweet orange genome, to provide a powerful tool for all future citrus improvement research.
The International Citrus Genome Consortium (ICGC), including groups from the United States, Brazil, France, Spain, and Italy, was established in 2003 to sequence a citrus genome and develop genomic tools to support citrus research worldwide (http://citrus_static.hivip.org/reports/2009/08/04/FCPRAC_ICGC_quarterly_report_8-09.pdf). This consortium has generated many
publicly available expressed sequence tags (EST) libraries, which collectively have over 550,000 ESTs (http://www.citrusgenome.ucr.edu).
In 2007, a 1.2X-coverage genome sequence of sweet orange was released by the US Department of Energy Joint Genome Institute (Talon and Gmitter, Jr., 2008). Using next-generation sequencing (454 of Roche Diagnostics), the ICGC has now reached over 30X coverage of the sweet orange genome coverage (FCPRAC, 2010). In addition, a number of citrus Bacteria Artificial Chromosome (BAC) libraries have been produced and are being curated at the Clemson University Genomics Institute. This effort should be redirected to take advantage of current technology and expanded to provide the underpinnings of a sweet orange conventional breeding system.
Discussion of the Merits of NI-4
Favoring rationale: Having the genome sequence of an organism in hand provides a tool with often unanticipated applications. Knowledge of the genome can be applied to citrus genetic improvement in general. Deciphering the citrus genome and additional genomic information (e.g., single nucleotide polymorphisms (SNPs) and ESTs) will provide scientists with a number of genomic tools that can be used to understand the genomic response of citrus in response to HLB, bacterial infection, and stress. Genomic-based research tools can support the creation of differential gene expression profiles to highlight differences between healthy and diseased citrus to aid in discovery of biomarkers applicable to both detection of diseased trees (Recommendation NI-2) and DNA markers for plant breeding. Genomic information and tools can support biotechnology and conventional approaches to support disease resistance in citrus. An example is knowledge of promoters and targeting sequences in support of transgenic citrus work. Progress on other plant genome projects suggests that progress on the citrus genome, and development of associated genetic tools, can be accelerated.
Sequencing of plant genomes, such as tomato and potato, have historically been conducted through large international consortia whose members can share the high costs and extensive computational investments and can manage a large-scale undertaking. The International Solanaceae Genome Project consortium involves 10 countries. By 2009, six years after the initiative was launched, one-third of the tomato genome had been sequenced. The Cacao Sequence project, an initiative launched in 2008, plans to sequence the cacao genome in under 5 years with sponsorship primarily from Mars, Inc., the USDA, and the participation of a few strategic partners. Declining sequencing costs and improvements in computational capacities may not warrant complex international collaborations for future sequencing projects. A global consortium may, in fact, delay the decision-making process and slow the pace of sequencing.
Early in 2009, a cucumber-sequencing project was begun by a group at the Beijing Genome Institute-Shenzhen with collaborators in several countries. This project employed a combination of traditional Sanger sequencing, which provides reads of ~700 base pairs (bp) and a state-of-the-art very high-throughput method (reads of 50 bp) in a hierarchical shotgun sequencing strategy to obtain a total of 26.5 billion bp of sequence. The sequencing effort produced 72-fold coverage, and 97.5 percent of the assembled and annotated sequence had a coverage of 10X or greater. The results revealed 26,682 genes and were published online in early November 2009 (Huang et al., 2009). The cucumber genome is 367 megabase pairs (Mbp); the sweet orange genome is 382 Mbp.
The accelerated effort recommended here could complement the efforts of the existing ICGC, as well as use the results and libraries it has generated. However, a new organizational
and funding structure, and possibly new partners to rapidly complete the citrus genome sequence are needed. The proposed strategy is to identify a small genome steering committee that represents the most important stakeholders and technology providers and a single funding source to complete the genome sequence. The steering committee needs to be equipped to respond rapidly to evolving sequencing technologies and assembly strategies should take into account complications introduced by citrus heterozygosity.
The overall project should develop high-density SNP markers and the assay platforms that will enable citrus breeders and scientists to accelerate genetic improvement strategies. The goal is to create an integrated project designed to complete, in short order, (1) the sequencing, assembly and annotation of a sweet orange genome using contemporary methods and meeting contemporary quality standards; (2) apply re-sequencing to document genomes of other citrus, including wild species; and (3) apply the information gained from genomic analyses to establish a workable conventional breeding system for sweet orange, including facile identification and evaluation of cross progeny.
Disfavoring rationale: The ICGC already consists of participants from several countries. If this recommendation is implemented, great care will be needed to ensure that the pace of sequencing the citrus genome is accelerated without the unintended effect of adding another layer of complexity to the existing organization of this initiative or reducing the enthusiasm of present participants. The estimated cost of sequencing the sweet orange genome is a few million dollars.
Example of currently or recently supported research projects and relevant results: The ICGC already is in progress.
Examples of possible future research projects: The end result of citrus sequencing efforts would be a large and very useful body of new information: the identities, structures, and in many instances the functions of the genes of sweet orange and eventually other citrus species and varieties. An example of the useful information expected would be a list of candidate phloem proteins (Huang et al., 2009), which would, in part, define the medium in which the phloem-dwelling CLas replicates in the citrus tree.
Related information and references: The genome sequences of about 7 plant species have been published. The general trend for each succeeding sequence has been to substantially lower costs and shorter time to completion. Except for the complication of heterozygosity of commercial citrus genomes, there is no reason that this trend would not benefit a citrus genome project.
Suggested support mechanism: Currently, the citrus genome is being conducted through an international consortium. The goal of this recommendation is to accelerate the genome sequencing, which may require a more effective and streamlined organizational structure.
Time to outcome: Finalizing the citrus genome is a high priority for research with significant benefits to the citrus industry; an aggressive short term strategy should be put in place as soon as possible to complete the sweet orange genome in less than 2 years.
Recommendation NI-5. Support development of model systems intended to reveal new approaches to HLB mitigation.
None of the elements of HLB are very amenable to controlled investigations of the underlying mechanisms of disease spread and development: not the disease agent, not the vector,
not the host, and not the environment. For these reasons, we are recommending that research proposals based on model systems rather than HLB-affected citrus itself receive serious consideration. Often research in model systems can yield new insights at relatively low cost andcan allow the application of high-throughput methods, not applicable to the original system, to test many compounds or genes for their potential as HLB mitigation agents.
Discussion of the Merits of NI-5
Favoring rationale: Model systems have allowed studies to be short-term and high-throughput, saving time and money. For example, it is conceivable that a citrus rootstock could be transformed to produce an anti-CLas protein that would move into and protect the scion. Candidate anti-CLas proteins almost certainly would be tested in a model plant system before moving to citrus. Model systems provide an opportunity to create a genuinely new and effective therapeutic—a silver bullet for HLB mitigation (see Recommendation L-2).
Disfavoring rationale: There are likely to be only a few model systems or test beds that will be relevant to HLB control.
Examples of currently or recently supported research projects and relevant results: A Rapid Screening Process for Chemical Control of Huanglongbing is a project being conducted by C. Powell, University of Florida. According to the proposal abstract: “This involves growing Can. Liberibacter-infected citrus and periwinkle in culture, and adding antibacterial molecules to the culture medium and assessing the effect on the bacteria by qPCR. This will allow testing hundreds of antibacterial compounds in a short period of time, and those compounds that are effective on HLB-suppression without phytotoxicity will be selected for field trial.”
Use of the tomato/potato psyllid, Bactericera cockerelli, vector for the zebra chip disease of potato, is also a valuable model system, in place of citrus trees and ACP. An herbaceous host has clear cultural advantages over perennial citrus and allows the use of virus vectors for high throughput testing of putative anti-CLas and anti-ACP genetic constructions. One of several projects underway is being conducted by B. Falk, University of California at Davis, who is developing anti-psyllid RNA interference (RNAi) (Recommendation NI-9) technology using tomato seedlings and B. cockerelli.
Example of possible future research projects: Chemical libraries could be screened for compounds capable of causing citrus to repel ACP. For example, genetically homogenous tomato seeds could be germinated in multi-well plates and then exposed to the compounds (a different chemical added to each well) in low micromolar concentrations. The treated seedlings could then be exposed to the tomato/potato psyllid and observed to see which are avoided by (or that attract) the psyllid.
Related information and references: In one study, Arabidopsis seedlings in liquid medium in 96-well plates were exposed to and subsequently infected by Pseudomonas syringae bacteria that induced bleaching of the cotyledons. Seedlings were exposed to micromolar concentrations of about 200 compounds from a chemical library prior to inoculation by P. syringae. A few sulfanilamide compounds dramatically reduced P. syringae-induced cotyledon bleaching and one compound reduced the P. syringae titer to about 10 percent of control value (Schreiber et al., 2008).
In another study, tomato seedling cotyledons were transformed with Agrobacterium rhizogenes to introduce an expression library of tomato cDNAs. The resulting library of tens of thousands of hairy roots was exposed to the apoptosis-inducing mycotoxin fumonisin B1. A few of the hairy roots remained white and growing. After several transfers of root segments in
fumonisin B1-containing medium, the cloned and selected cDNA sequences were recovered by PCR (Harvey et al., 2008). Subsequently, the corresponding transgenes have demonstrated significant sparing effects against invasion by specific apoptosis-inducing pathogens.
Suggested support mechanism: Proposals on model systems require expert evaluation both for the feasibility of the model system and for its appropriateness to the HLB problem, favoring a competitive grants approach for providing research support.
Time to outcome: Mid-term to long-term.
Recommendation NI-6. Exploit the CLas genome sequence for new strategies of HLB mitigation.
The CLas genome sequence was recently completed and annotated (Duan et al., 2009; Tyler et al., 2009). Implementation of recommendation NI-6 is intended to identify CLas targets for future HLB mitigation technologies.
Discussion of the Merits of NI-6
Favoring rationale: Bioinformatics can produce what is, in effect, a highly educated guess (annotation) about the functions of an individual gene from the nucleotide sequence of the gene alone. The available annotated CLas genomic sequence (Duan et al., 2009) revealed the presence of genes encoding several Type I secretion systems but no Type III secretion system and no cell-wall degrading enzymes. Knowing what genes are not present is as informative as the observed gene complement; for example, for use in the design of synthetic growth media for CLas or for insight into the offensive and defensive tactics that may be taken by CLas in its plant or insect host. Transfer of CLas genes to Escherichia coli or other bacterium suited to experimental manipulation or transient expression of CLas genes by means of bacterial- or viral- based molecular vectors in plant or insect hosts can reveal the functions of CLas genes. Knowing the functions of specific CLas genes can direct approaches to interfering with essential gene function, in effect controlling the bacterium.
Disfavoring rationale: Although the annotated CLas genome sequence is a valuable information source for identifying gene products as potential targets, developing and applying test systems to verify gene product function and susceptibility of the potential target to interference are more difficult and expensive.
Example of currently or recently supported research projects and relevant results: Several research groups are investigating various CLas genes for gene products that may be susceptible to interdiction by chemical or genetic approaches.
Suggested support mechanism: A peer-review-based competitive grants program would be the best mechanism for identifying projects likely to advance mitigation of HLB.
Time to outcome: Demonstration of proof of principle, (e.g., by reducing CLas growth in a model system) could be achieved in months or a few years. The actual application to citrus would be a longer-term process.
Examples of possible future research projects: The CRDF published a request for proposals intended “to test new methods of disease control made possible using the bacterial genome sequence recently published.” It is likely that awards for new projects in this area will be made in early 2010.
Other sections of this report containing related information: Chapter 3, p. 68 (CLas genomics).
Recommendation NI-7. Develop alternative ACP management strategies.
As is widely recognized, thus far only the application of conventional insecticides has succeeded in reducing ACP populations to the point that HLB mitigation is demonstrated. However, dependence on traditional insecticides has potentially adverse consequences to sustainable orchard practice. In the future, ACP-resistant commercial citrus lines created using RNA inteference (RNAi) or other genetic means (see Recommendations NI-9 and L-1) have very good potential to provide HLB mitigation. However, in the near and intermediate terms there will be a need not only for new chemical insecticides, adjuvants, and better surveillance and application methods and modified cultural practices (Recommendation NI-1), but also for new biochemical methods of suppressing ACP populations. Non-insecticidal ACP life-cycle disrupters and chemical and biological repellents and/or attractants have the potential to suppress ACP populations or CLas transmission while having minimal effects on natural enemies. ACP is the target of some natural enemies in Florida. It is worthwhile to use ACP control options that tend to preserve ACP enemies. However, for systems such as HLB, in which the insect vector transmits a pathogen which causes rapid crop loss, biological control of the insect vector alone has not resulted in significant crop protection. Therefore, we believe that at this time it will be prudent not to direct significant resources to ACP biological control strategies but rather to opportunities for other non-insecticidal chemical management.
Recommendation NI-7a. Investigate ACP-citrus interactions to develop non-insecticidal and possibly highly ACP-specific compounds capable of interfering with the ACP life cycle or its interaction with Clas.
ACP is well adapted to and completes its life cycle on citrus, which in general ACP prefers to other plant hosts (Halbert and Manjunath, 2004). This adaptation suggests that ACP responds to specific characteristics of citrus such as plant compounds that act as ovipositional and feeding cues. Similarly, the intimate contact between ACP and citrus during feeding may involve ACP countermeasures against defenses that citrus would be expected to mount against invading insects. Results derived from research under Recommendation NI-2 (test plots) and NI-11 (the ACP genome sequence) could inform a search for compounds involved in chemical communications between ACP and citrus, and for compounds capable of interfering with such communications, e.g., with the ability of ACP to locate and lay eggs on suitable sites on the host or to protect itself against citrus insect defense. Other strategies could be aimed at discovering compounds that disrupt processes of ACP development, mating, and ability to transmit the bacterium. Obviously, compounds of the types described here, although much more difficult to identify than conventional insecticidal compounds, have the potential to be far more ACP-specific and therefore may prove more sustainable in their use than conventional insecticides.
Recommendation NI-7b. Develop repellents/deterrents and attractants for ACP.
If an appropriate system with a highly attractive plant species can be developed, trap cropping could be used to lure ACP away from orchard citrus trees and concentrate them onto plant species that can withstand an infestation and allow large numbers of ACP to be killed with minimal insecticide application. A few very successful trap crop programs have been
implemented on a commercial scale (Shelton and Badenes-Perez, 2006). However, because the impact of ACP is by its transmission of CLas, achieving effective disease mitigation through use of repellents and attractants is far more difficult for HLB. If repellent/deterrent chemicals are identified, it is possible that they could be applied to citrus to drive ACP away. Likewise, if attractants are applied to other plant species that are not suitable hosts of ACP or HLB, they could be used to protect citrus.
Discussion of the Merits of NI-7
Favoring rationale: Reduction in the use of conventional pesticides continues to be an important trend in modern agriculture, which runs counter to continued heavy reliance on traditional insecticides for management of ACP. The reasons for this trend are several, including reduction in production costs for the grower, benefits to the environment, real and presumed hazards of pesticides to consumers and farm workers, and marketing strategies (e.g., organic food and bio-based labeling). In cases where production practices do not allow the use of traditional insecticides (e.g., organic production), alternative strategies will be especially important. The current strategy for management of ACP relies on an aggressive program of soil-and foliar-applied insecticides. History has taught entomologists that sole reliance on insecticide-based strategies will not be sustainable in the long run, or oftentimes not even in the short run. Other management strategies should be pursued as part of an overall integrated pest management (IPM) approach. Although little is presently known about specific interactions among ACP, CLas, the plant host and the environment, results from other vector systems suggests that information on these topics could assist in HLB mitigation.
Disfavoring rationale: Many, if not all, alternative chemical strategies for ACP management are only in the conceptual stage or in very early phases of development. There does not appear to be any alternative strategy that is ready for implementation at this time and can match the effectiveness of current insecticide application regimes. This observation suggests that the search for such strategies will not be easy. With the current HLB crisis facing the citrus industry, it would be unwise to move rapidly from reliance on traditional insecticides to approaches with incompletely explored capabilities and potential problems.
Examples of currently or recently supported research projects and relevant results: There are many currently funded projects exploring alternative management practices for ACP. These include three projects using RNAi (which are likely to be long term to application), one project investigating the ACP transcriptome, three projects using some form of trap cropping to disrupt the ability of ACP to colonize citrus, and other projects that are focusing on enhancing biological control of ACP.
Examples of possible future research projects: New strategies may arise from an understanding of the ACP genome (see Recommendation NI-11).
Related information and references: Whatever strategies are used to manage ACP, they should be used in conjunction with a more complete understanding of the biology/ecology of ACP as well as the landscape in which the strategies are deployed to delay the evolution of resistance to a particular tactic (e.g., Storer et al., 2001). In a perennial cropping system such as citrus, which incurs a large initial investment, durability of the system is essential for longer term sustainability. A region-wise set of management strategies (Recommendation O-1) is vital to the success of such a program.
Suggested support mechanism: Projects currently testing alternative management strategies should be continued and new projects should be supported on a competitive basis with proposals evaluated on scientific merit and potential benefit.
Time to outcome: Research in progress at this time may yield replacements or partial substitutes for current insecticide applications, but it is more likely that effective alternative ACP suppression approaches will be available only after years of research and development. However, incorporation of traditional insecticides into more effective and sustainable IPM programs offers the possibility of near-term improvements with long-term benefits.
Other sections of this report containing related information: Chapter 3, pp. 72–76 (reducing ACP access to citrus).
Recommendation NI-8. Support small-scale studies on the feasibility of alternative horticultural systems that may lead to citrus production systems suited to endemic HLB.
If shortages drive prices higher, some cultural practices now deemed economically unfeasible may become viable. Recommendation NI-8 is intended to encourage the exploration of new citrus production systems such as high-density plantings and screen houses. The advanced citrus production systems may be able to compress and enhance the citrus production cycle so that economic return can be realized in fewer years (Hutton and Cullis, 1981). Early economic fruit production could be reached in as little as three years, thus achieving an early return on investment.
Discussion of the Merits of NI-8
Favoring rationale: Most advanced production systems involve high-density plantings and intensive management. The advantages of these systems are that production begins earlier in the life of the grove, yields are higher, and fruit from shorter trees would be easier to harvest. Grove life would be shorter, but close spacings would allow cost recovery and profitable production before HLB eventually took over the grove, which would then be replaced.
Disfavoring rationale: The disadvantages of advanced production systems are the high initial cost of orchard establishment and the more intense management required. These systems can probably function, but the economic viability of the systems in Florida and the manageability of the system over the long term will need to be investigated. These advanced production systems seem better adapted to production of fresh fruit and less appropriate for large-scale production of processing oranges. Many groves were planted at high density in Florida following the freezes in the 1980s. Early production was much higher than at conventional spacings, but when no freezes occurred in subsequent years, groves became unmanageable and growers reverted to more conventional tree spacings. Such advanced plantings would still have to be managed by removal of HLB-affected trees and application of insecticides for psyllid control. To be viable, these plantings would need to be located at some distance from sources of inoculum, (e.g. by conversion of pasture to citrus orchard).
Example of currently or recently supported research projects and relevant results: Plantings were made in 2006 at the Southwest Florida Research and Education Center comparing tree densities of 545, 198, and 151 per acre, with early and late oranges on different rootstocks. These plantings are being evaluated for yield, production costs and other parameters.
One promising high density planting system, the Open Hydroponics System (OHS), was developed in South Africa (Katz, 2008). For applications in Florida, OHS requires adaptation to Florida’s summer rainy season and sandy soil. High density plantings using the OHS are being
evaluated by Arapaho Farm Management, Inc. (http://www.arapahocitrus.com/ohs.html) on Florida’s east coast.
A currently supported project (principal investigator Schuman) is concerned with intensively managed citrus production systems for early high yields and vegetative flush control in the presence of HLB.
Examples of possible future research projects: Flying Dragon trifoliate is the only commercially used rootstock that reduces tree size substantially and may be useful in these systems. Dwarfing of citrus trees by inoculation with viroids has been studied extensively in Australia and elsewhere (Hutton et al., 2000; Semancik, 2003; Hardy et al., 2007). Viroid species-rootstock combinations could be investigated as a means to reduce tree size to allow for closer tree spacing and earlier production and to adapt this approach for use in OHS.
Related information and references: Most of the current focus of alternative horticultural systems for citrus is on hydroponic-like systems for reducing the size of the tree root ball (Aki et al., 2008; Land and Water Australia, 2008) and on reducing tree size (Hutton and Cullis, 1981; Hutton et al., 2000; Semancik, 2003; Hardy et al., 2007)
Suggested support mechanism: Investigator-initiated proposals.
Time to outcome: Many years of investigation and grower experience would be needed before such systems could be recommended and widely utilized.
Recommendation NI-9. Support demonstration of RNA interference effects for possible suppression of ACP.
More than a decade ago, the phenomenon of RNAi was demonstrated in several phyla and now is recognized as a general and fundamental regulatory mechanism of biology. In the application of RNAi to negatively affect ACP feeding on citrus, the most feasible approach would involve transformation of citrus to accumulate, in phloem tissue, double-stranded RNA (dsRNA) corresponding in nucleotide sequence to an essential ACP gene. Under this scenario, the ACP RNAi system would use the dsRNA to interfere with the accumulation of ACP messenger RNA corresponding to the essential gene, resulting in mortality, morbidity or fecundity reduction of ACP. It is possible that protection could be achieved by transforming citrus rootstock to which untransformed scion would be grafted. Having available sequences of ACP genes (see Recommendation NI-11) will contribute to this approach.
Developing methods for effectively delivering RNAi-active molecules to ACP presents the greatest challenge to the application of RNAi technology. Experimentally, dsRNA administration to insects for initiation of RNAi is usually by injection (Begum et al., 2009) or by topical application (Pridgeon et al., 2008), but practical application would be by ingestion of dsRNA from phloem. RNAi activity from ingested dsRNA has been demonstrated in several systems (Price and Gatehouse, 2008; Zhou et al., 2008). Both predominantly single-stranded and highly structured phloem-located RNA molecules, their long distance transport in the plant, and in some instances their functions, have been demonstrated (Kalantidis et al., 2008; Banerjee et al., 2009; Turgeon and Wolf, 2009), so potential sources of RNA signals for transgene-mediated dsRNA delivery to phloem tissue are known, as are phloem-specific promoters (Chakraborti et al., 2009; Srivastava et al., 2009). Primary cell cultures from ACP have been reported (Marutani-Hert et al., 2009a). ACP cells in culture could provide a test bed for selection of sequences for their effectiveness against ACP.
The goal of Recommendation NI-9 is to establish proof-of-concept for this potentially new approach to ACP management. Fortunately, proof-of-concept can be established without use of transgenic citrus by taking advantage of transient expression and detached leaf uptake approaches.
Discussion of the Merits of NI-9
Favoring rationale: As is indicated above, citrus cultivars showing resistance to CLas and ACP are likely to be a critical part of effective management for HLB in the long term. Currently, there is no obvious path to creating ACP-resistant citrus by conventional breeding, and the RNAi approach could provide one form of ACP-resistant citrus.
Disfavoring rationale: Insect species vary in their susceptibility to the RNAi approach, and the susceptibility of ACP is unknown. Where an effect of dsRNA on the insect has been demonstrated, the concentrations of dsRNA that have been supplied have typically been close to 1 microgram per milliliter (1g/mL). Presumably only much lower concentrations of dsRNA could be achieved in phloem fluid of transgenic citrus. There is no established method for transforming any plant to generate dsRNA in its phloem sieve tubes, so this technology would be needed before RNAi can confer ACP-specific insecticidal capability. Artificial diet-feeding of psyllids, which would be convenient for the testing of a variety of dsRNA constructions, is not readily accomplished for ACP.
Example of currently or recently supported research projects and relevant results: Three currently supported HLB research projects are aimed at identifying sensitive target sequences and administering dsRNA to a psyllid for the purpose of showing detrimental changes in phenotype. One of these projects is developing a method that will allow many different RNA sequences to be tested by a transient expression approach, which could uncover target sequences for which ACP would show a strong sensitivity, reducing the need for high level dsRNA accumulation in the transgenic plant.
Examples of possible future research projects: Investigations of systems for delivery of transgenic RNA (and proteins) to phloem sieve tubes would benefit RNAi work as well as other aspects of citrus improvement.
Related information and references: Demonstration of an RNAi action in insects was accomplished first by injection of dsRNA. However, Araujo et al. (2006) prepared dsRNA corresponding to a 548-bp segment of the Rhodnius prolixus (Chagas disease vector, Hemiptera) salivary nitrophorin-2 gene and were able to show a phenotype (reduction in blood coagulation time to about one-quarter of control values) both by injection and by feeding of the dsRNA, at a concentration of 1 g/mL. RNAi action has been demonstrated in aphids (Mutti et al., 2006; Mutti et al., 2008) and whiteflies (Ghanim et al., 2007). RNAi has also been reported to be active against coleopteran and lepidopteran insects fed dsRNA in their diets (Baum et al., 2007; Mao et al., 2007).
Suggested support mechanism: The extensive basic research needed to provide proof-of-concept for plant-generated dsRNA with anti-psyllid RNAi capability will be best supported by competitively awarded grants.
Time to outcome: Demonstration of deleterious action of injected dsRNA against intact psyllids likely will be reported shortly. Similar action by dsRNA artificially introduced into plant phloem should follow within a year or two. Protection based on dsRNA synthesis and phloem secretion in a transgenic plant, if sufficient dsRNA can be generated, will likely require years of research.
Recommendation NI-10. Develop in vitro culture techniques for CLas to facilitate experimental manipulation of the bacterium for insights into gene function.
The short-term culture of CLas in vitro has been reported, but culture of CLas over multiple transfers apparently has not been accomplished. Availability of in vitro culture technology for CLas as a general laboratory method could facilitate the finding of new insights into the physiology of this organism and possibly identify new points of vulnerability for control strategies.
Discussion of the Merits of NI-10
Favoring rationale: If the in vitro culturing of CLas were to become sufficiently facile, CLas could be considered to have been “isolated” and Koch’s postulates could be completed, with CLas (presumably at that point becoming Las, no longer “Candidatus”) identified unequivocally as the causal agent of HLB. Additionally, creating pure cultures of the bacterium in vitro should lead to improved understanding of this organism and to its genetic transformation, which would allow production of green fluorescent protein-expressing or otherwise marked strains and to the testing of individual genes for their role in citrus infection and virulence, ACP infection, and other functions of the bacterium.
Disfavoring rationale: This recommendation is not as strongly supported as it might have been just a few years ago because modern biology has provided enhanced capabilities for analysis of CLas characteristics in its natural environment, i.e., the infected plant or insect. Such analyses are likely to provide relevant information about CLas that would not readily be obtained from plate or liquid cultures.
Example of currently or recently supported research projects and relevant results: There are at least five currently funded projects aimed at culturing CLas, taking different approaches but usually attempting to mimic conditions of plant phloem or the insect body.
Examples of possible future research projects: It is possible that variations on the approaches that have given limited increase of CLas in vitro could provide a starting point to which many variations could be applied with the goal of supporting unlimited increase of CLas in culture. Availability of the CLas genome sequence may yield enough clues about the bacterium’s metabolism to direct the incorporation of specific compounds into trial synthetic media.
Related information and references: Limited in vitro colony transfer of CLas has been reported using a solid medium containing citrus vein extract (Sechler et al., 2009).
Suggested support mechanism: Either a standard research grant approach or an incentive prize could be considered.
Time to outcome: Considerable funds and effort have been invested in attempts to culture CLas. The time necessary to achieve success is uncertain.
Recommendation NI-11. Sequence, assemble and annotate the ACP genome to provide a basis for new approaches to ACP management.
Recommendation NI-11 is intended to create information on which to base future strategies for ACP suppression or interference of CLas transmission by ACP (Recommendation NI-7).
Discussion of the Merits of NI-11
Favoring rationale: Where the complete genome of an animal is available, whether it be of humans or a small invertebrate (Opperman et al., 2008), benefit has been derived almost immediately from the investment in the form of genes as targets for disease amelioration or life cycle functional interference, in addition to new understanding of the organism as a whole. Thus far, a number of insect genome sequences are known, including the fruitfly, Drosophila melanogaster; honey bee, Apis mellifera; malarial mosquito, Anopheles gambiae; red flour beetle, Tribolium castaneum; and silkworm, Bombyx mori (Hart and Grosberg, 2009). The genome of the pea aphid, Acyrthosiphon pisum is also available (IAGC, 2010). For ACP, genome information could help identify targets likely to be accessible in gut cells and sensitive to interference by RNAi approaches (Recommendation NI-9), targets for chemical control and prophylactics or therapeutics, and factors that may be involved in nymph survival or ability to support CLas in ACP (Recommendation NI-7). The first determination of the haploid genome size for a phytophagous psyllid, Pachypsylla venusta, the hackberry petiole gall psyllid, was reported (Nakabachi et al., 2009) as 724 million base pairs, suggesting that sequencing the ACP genome could be readily accomplished using standard techniques.
Disfavoring rationale: Genome projects require significant initial investment and continuous supervision by expert management to coordinate selection of the source of genomic DNA, preparation of various types of genomic libraries, management of samples, the efforts of sequencing facilities, and quality controls. Significant bioinformatics capabilities for assembling and annotating the sequence are also required. As was noted, the complete genome sequence of an insect pest of plants has been accomplished only for the pea aphid, and as yet, there has been no demonstration of suppression of such insects based on genome information.
Example of currently or recently supported research projects and relevant results: A psyllid genomics consortium (http://www.ars.usda.gov/pandp/people/people.htm?personid=11768) has been started, and several datasets derived from ACP sequences are available at the National Center for Biotechnology Information (NCBI) (website www.ncbi.nlm.nih.gov).
Examples of possible future research projects: The value of a genome sequence lies in the ability of researchers to predict functions of genes from sequence alone and to identify genes that may be targets for manipulation of the organism—in the case of ACP, suppression. The genome sequence of a human parasitic nematode has been subject to bioinformatics analysis using the very extensively studied, free-living model nematode, Caenorhabditis elegans, as the reference for gene function information. The analysis revealed about 600 drug target candidates (Kumar et al., 2007). Similarly, the ACP genome sequence could be “mined” for insecticide targets, RNAi targets, and new types of targets that might be revealed in the analysis, using the very extensively studied model insect D. melanogaster (www.fruitfly.org) and the recently sequenced pea aphid genome (http://www.hgsc.bcm.tmc.edu/project-species-i-Pea%20Aphid.hgsc) as reference sequences.
Related information and references: An EST library of ACP has been prepared representing about one-fifth of the likely transcripts of the organism (Hunter et al., 2009), and sequences suggesting the presence of a virus of ACP were detected (Marutani-Hert et al., 2009b).
Suggested support mechanism: Presuming that there is more than one group seeking support for genome sequencing, funds should be awarded competitively.
Time to outcome: Significant sequence information, and perhaps even the entire genome sequence, could be accomplished in less than two years using current high-throughput methods.
Other sections of this report containing related information: Appendix K.
RECOMMENDATIONS FOR RESEARCH AND TECHNOLOGY WITH THE POTENTIAL TO ADVANCE HUANGLONGBING MITIGATION IN THE LONG-TERM
Given the urgency of the HLB situation in Florida, most investments in research and management are necessarily near-term. However, continuing investments in research aimed at longer term solutions remains important. In addition to the four long-term recommendations below, completing the citrus genome sequences and exploiting them to create usable breeding systems for commercial citrus (Recommendation NI-4) should be a priority. The genome sequence, when combined with high-throughput SNP genotyping and other additional information, can facilitate improvement of Florida citrus varieties.
TABLE 4-4 Long-Term Recommendations
Support development of transgenic HLB-resistant and ACP-resistant citrus.
Support development and testing of bactericides, therapeutics or SAR activators.
Support analysis of ACP behavior, ACP-plant interactions and ecology to enhance the knowledge base available for new ACP management strategies.
Explore possible control strategies based on release of modified psyllid males.
Recommendation L-1. Support development of transgenic HLB-resistant and ACP-resistant citrus, including creating suitable anti-CLas and anti-ACP genes.
It is generally agreed that CLas- or ACP-resistant citrus would provide the ideal long- term management tool for HLB. Currently, the most promising path to developing resistant citrus involves genetic engineering (GE). Advances in GE approaches include techniques to utilize mature tissue, to bypass the juvenile stage and accelerate the transformation and regeneration process (Cervera et al., 1998). Other research groups are exploring transgenes that may confer resistance to pathogens other than CLas or greater tolerance to low temperatures. To expedite rapid screening of candidate trait genes, Citrus tristeza virus (CTV)-derived vectors are available for stable transient expression in citrus (Folimonova et al., 2007) and can be used to provide a testbed for gene constructions designed to have anti-CLas or anti-ACP activity.
Although there are non-citrus plants that demonstrate resistance to CLas or ACP, such plants are not likely to be sources of genes useful in citrus because of inter-species incompatibility and difficulties in isolating such genes. Sequences encoding anti-CLas transgenes are more likely to be anti-microbial peptides and proteins (AMPs) of animal, plant, microbial or bacteriophage origin (Canny and Levy, 2008; Conesa et al., 2009; Soehnlein, 2009; Mao et al, 2010). Similarly, there are many possible sources for sequences that could encode anti-ACP proteins (Sauvion et
al., 2004; Staniscuaski et al., 2005; Down et al., 2006; Gonzalez-Zamora et al., 2007; Follmer, 2008), including venom components, lectins, bacterial spore proteins, protease inhibitors, and vitamin-binding proteins.
From a practical standpoint, GE strategies should consider traits that can be delivered via rootstocks that can be deployed across a number of scions. Several rootstocks are in use in Florida, and each rootstock would need to be transformed independently unless conventional genetic crossing is available. Nevertheless, incorporation of the transgene into a few rootstock lines would be considerably less expensive than transforming many scion lines. Of course, replacement of existing trees is an additional significant expense and an expense that will likely continue well into the future. In-arch grafting would allow rootstock substitution for existing trees, reducing the need for replacement. An orchard citrus tree may have a one-hundred year life span, demanding durable resistance. Thus, strategies for constructing transgenic, disease-resistant citrus should involve the pyramiding of transgenes with different mechanisms of action in order to minimize the chances of CLas or ACP overcoming resistance.
As is described in Chapter 3, CTV vectors have shown exceptional longevity for expression of foreign genes in citrus, so CTV vectors may provide benefits beyond serving as a testbed for candidate transgenes. An effective CTV construction conferring anti-CLas or anti-ACP biology or transmission ability could be introduced into existing orchard trees by conventional grafting techniques, providing protection against HLB and even therapeutic effects without tree replacement. Thus, CTV-vector-mediated HLB mitigation strategies warrant serious attention. In addition, the research strategies adopted, whether using conventional transformation or CTV, should be designed with full consideration of achieving the necessary downstream regulatory approvals and intellectual property freedom-to-operate.
Discussion of the Merits of L-1
Favoring rationale: Genetically engineered resistance, whether by conventional transformation or introduction by CTV vector, has the greatest potential of available technologies for long-term HLB disease management (Singh and Rajam, 2009). For conventional transformation, creating high-throughput transformation protocols with an accelerated regeneration timeframe of transgenic citrus is a critical first step. Recent advances in citrus transformation technology and the availability of potential resistance-conferring transgenes will allow more rapid progress than would have been possible even five years ago.
Disfavoring rationale: A realistic evaluation of GE strategies suggests that although there is promising research in this area, there are significant technical challenges that will need to be overcome. The need, particularly in the long run, for durable resistance requires that multiple transgenes be developed to provide pyramided protection against both CLas and ACP. Creating multiple resistance transgenes will be technologically very demanding.
Any new transgenic crop is subject to extensive regulatory procedures before commercialization can be approved, requiring significant investments of money and time. Even if a transgenic citrus line is approved for commercialization, grower and public acceptance remains uncertain.
Example of currently or recently supported research projects and relevant results: In 2008–09, about $2.1 million dollars were invested in citrus research focused on transgenic and viral/bacterial vector mediation of citrus resistance to HLB. See projects listed in Appendix J, transgenic and viral/bacterial vector mediation of citrus resistance to HLB.
Examples of possible future research projects: Future research projects stemming from this recommendation include an assessment of the feasibility of using transgenic rootstock to deliver disease resistance to a wide variety of scions. Research strategies will require testing promoters and signal sequences that mobilize the transgenic molecules across the graft union. Both proteins and molecular signals of other types have been demonstrated to traverse a graft union (Golecki et al., 1998; Prassinos et al., 2009), supporting the concept of rootstock-delivered anti-CLas or anti-ACP molecules.
There is no regulatory precedent for a system in which resistance is conferred on the scion from a transgenic rootstock. As is likely for any other new regulatory situation, rootstock protection may have special requirements for field trials and data collection, which will need funding.
Since intellectual property is another key element in developing transgenic citrus, it may be important to fund future research projects that validate technologies with freedom-to-operate that may be used as foundation technologies in citrus bioengineering. Here, we anticipate it may be necessary to support research projects that analyze the international legal issues surrounding the key transformation technologies and other projects that test the functionality of these technologies in citrus; for example, plant selection genes, Agrobacterium-mediated transformation, plant transformation vectors and transcription promoters.
Another future research project should consider the development and/or deployment of marker-free transformation technologies. There is debate within the international community about whether marker-free plants will offer consumer acceptance advantages or facilitate regulatory registration, but there is emerging regulatory resistance to allowing antibiotic resistance genes in Europe.
Suggested support mechanism: It is clear that research in genetic engineering of citrus has a long horizon and will require a multi-disciplinary team approach with funding to laboratories working on the evaluation of resistance gene strategies, developing efficient transformation protocols, field-testing transgenic trees, developing regulatory information and ensuring intellectual property freedom-to-operate. To ensure coordination of these activities, there should be a funding program with a guidance board dedicated to identifying and funding a consortium of laboratories to quickly advance the development and deployment of transgenic citrus trees.
Time to outcome: GE strategies are recognized as one of the areas with greatest potential and should be supported immediately. This long-term objective probably will require 10–15 years of research investment and regulatory approval and public education efforts, although if current field trials were successful and accelerated approval processes were taken up, commercialization of CLas- or ACP-resistant citrus could occur in less time. Implementing a collective research effort that also focuses on intellectual property and regulatory compliance will accelerate development, field testing, and registration.
Other sections of this report containing related information: Chapter 3, pp. 78–83 (transgenic citrus, anti-CLas genes), and pp. 84 (CTV vectors).
Recommendation L-2. Support development and testing of bactericides and other therapeutics or activators of systemic acquired resistance for control of CLas.
Development of a bactericide or some other curative product that could be applied for control of HLB would provide many advantages. Tree removal would not be necessary and control of psyllids might not need to be so rigorous. However, there are no systemic bactericides, except for antibiotics, that have been registered for use on citrus or any other crop. The bactericides that
currently exist are copper products that have been used for centuries and antibiotics that were developed in the 1940s. Manufacturers of agrichemicals have invested only to a limited extent in the development of such products and there are no potentially useful products of this type on the horizon. Activators of systemic acquired resistance (SAR), such as salicylic acid and phosphorous acid, are available on the market and some are registered for use on citrus. These products have generally provided only a low level of control of bacterial diseases. The systemic insecticide, imidacloprid, currently used for psyllid control on citrus, has shown some SAR activity for control of citrus canker and may be of some value for HLB mitigation.
Discussion of the Merits of L-2
Favoring rationale: Bactericides could provide a solution like no other: curative action effective on the already CLas-infected tree, eliminating the need for other control measures for HLB. If such products could be applied by foliar sprays, conventional techniques familiar to growers could be used for treatments.
Disfavoring rationale: No currently available product is likely to provide the potency, at an affordable price, needed to make other control measures unnecessary. Even if symptoms were reduced to an acceptable level, CLas would probably persist in the trees and continue to be sources of inoculum for infection of other trees. Also, the bacterium might be able to develop resistance to any product applied, so this control measure might not endure. Tree injection of antibiotics was used for a time for control of HLB in South Africa, but control was not complete, application was difficult, and the practice was eventually discontinued. SAR products would be prophylactic and are unlikely to provide the level of control necessary to avoid other control measures. A practical product would need to be persistent in effect and be of relatively low cost.
Example of currently or recently supported research projects and relevant results: Current HLB projects include four on SAR (Graham, Lu, Rouse, and Stansly) and one on screening compounds for anti-CLas activity (Powell).
Examples of possible future research projects: High-throughput model systems (see Recommendation NI-5) have the potential to sort through many hundreds of compounds to identify those with prophylactic or therapeutic capabilities but with minimal adverse consequences to the plant.
Related information and references: SAR is a widely recognized phenomenon but one that has found only limited application in plant protection (Durrant and Dong, 2004; Francis et al., 2009). Similarly, there have been few applications of therapeutics in the control of citrus diseases (Schwarz and von Vuuren, 1971; Buitendag and von Broembsen, 1993; Layden, 2009) or any plant disease.
Suggested support mechanism: Investigator-initiated proposals to a competitive grants agency.
Time to outcome: Some products available now could prove helpful short-term, but development of highly effective products would likely require many years of research.
Other sections of this report containing related information: Chapter 3, pp. 83, 86 (systemic acquired resistance); Chapter 3, p.87 (model systems); Appendix J, (citrus response to infection); Appendix K (systemic acquired resistance genes in transgenic grapefruit).
Recommendation L-3. Support analysis of ACP behavior, ACP-plant interactions and ecology to enhance the knowledge base available for new ACP management strategies.
Control of ACP by currently available conventional insecticides will not likely be sufficient for long term mitigation of HLB. Newer insecticides will be helpful, but their use will be constrained by costs of the insecticides and their applications, uneven distribution of the insecticide within trees, the evolution of insecticide resistance and damage to non-target organisms and the environment. To overcome those barriers, a better understanding of the CLas/ACP/citrus-HLB system is needed. This recommendation is aimed at extending and fortifying the most promising observations obtained under Recommendation NI-1c, and at bringing them to practical application through the development of a mathematical model of the plant-vector-pathogen complex.
Mathematical models that incorporate data such as those expected to be generated under Recommendation NI-1c (behavior of ACP adults and nymphs; interactions of ACP, CLas, citrus trees, other organisms and the environment) provide a tool through which the results of behavioral ecology can be applied to disease mitigation (Gonzalez-Zamora et al., 2007; Follmer, 2008; Mitchell et al., 2009; Cunniffe and Gilligan, 2009). Although information on ACP behavioral ecology that falls short of what may be needed for a model will still be instructive for ACP management in the near-term, the construction of a mathematical model for the CLas/ACP/citrus-HLB system should be a long-term goal of HLB research. It is recognized that the complexity of pathogen transmission by an insect vector will make the task very challenging.
Discussion of the Merits of L-3
Favoring rationale: Compared with other phytophagous hemipterans, relatively little is known about the behavior and ecology of psyllids, including ACP, so what new information may be collected will expand the knowledge base significantly with likely application to HLB mitigation.
Disfavoring rationale: Translating results from research on ACP behavioral ecology into ACP management and HLB mitigation, including the construction of an appropriate mathematical model, will be difficult and likely time consuming.
Example of currently or recently supported research projects and relevant results: No studies targeted on the behavioral ecology of the immature stages of ACP are funded. The only references regarding nymphal behavior are anecdotes published in extension publications (Grafton-Cardwell et al., 2006; Rogers and Stansly, 2006).
Related information and references: Several references examine behavioral aspects of psyllids and other small piercing and sucking herbivorous insects (Dawson et al., 1990; Novak, 1994; Cocroft, 2001; Luft et al., 2001; Grafton-Cardwell et al., 2006; Rogers and Stansly, 2006; Guedot et al., 2008; Wenninger et al., 2008; Wenninger et al., 2009a; Wenninger et al., 2009b).
Suggested support mechanism: Competitive grants seem most appropriate. Several investigators are currently funded to study various aspects of the biology, ecology and behavior of adult ACP. It is suggested that these researchers meet, discuss the merits of the above recommendations, and expand their studies with existing funding to include the behavioral ecology of immature ACP.
Time to outcome: New information about the behavior of ACP immature and mature forms can be obtained and exploited within a year or two. For example, newly discovered attractants, of psyllid or plant origin, could be included in psyllid traps to improve trapping efficiency.
Conversely, discovered repellents could be used alone or in combination with insecticides to better manage psyllids.
Other sections of this report containing related information: Chapter 3, p. 76–78 (ACP behavioral ecology).
Recommendation L-4. Explore possible psyllid control strategies based on release of modified psyllid males.
The sterile insect technique (SIT) (the release of x-ray-irradiated males), has proven to be successful in several management strategies for agriculturally important pests. Newer techniques, which exploit genetically transformed insects, produce populations of males that are not debilitated by irradiation and therefore can compete with wild males. Recommendation L-4 explores the potential for the newer technologies to suppress ACP using these techniques.
Discussion of the Merits of L-4
Favoring rationale: The psyllid is a high-priority target for HLB management because it is an essential link in the infection cycle and is potentially vulnerable to a variety of control measures. In SIT, the target insect is raised on a mass scale and males are segregated, irradiated to induce sterility, and released into the area of the target insect population to compete with wild males for the available females. Separating the males is not trivial for many insect species. A recently developed alternative technology employs insects genetically transformed for a female lethal gene. This gene is suppressed by a component of the insect diet during rearing, that component being withheld at the last generation before release to yield a male-only population not capable of fathering females (Fu et al., 2007).
Disfavoring rationale: Compared to nymphs, adults acquire the HLB bacterium at a low efficiency, but not zero, so large releases of psyllids could increase HLB transmission marginally. Psyllids can reproduce to very high populations on some trees. Psyllid females mate serially. Therefore, the psyllid population would need to be reduced significantly before release of modified males, and, even so, large releases would likely be required to be effective in reducing the population in the next generation. Raising psyllids on a large scale at low cost will almost certainly require an artificial diet. No one has succeeded in rearing psyllids from egg to adult on a synthetic diet, much less the industrial-scale rearing that would be required to raise psyllid males for release. To date, SIT has not been developed for a phytophagous hemipteran.
Example of possible future research projects: (a) develop an artificial diet for rearing ACP from egg to adult and then develop procedures for producing psyllids on a large scale; (b) isolate female-specific gene or genes from ACP and prepare constructs for conditional expression of a lethal protein under control of the corresponding female-specific promoter; (c) rear the transgenic ACP on the artificial diet under conditions (e.g., diet component) that suppresses expression of the female-lethal gene; (d) prepare males for release by altering conditions to promote expression of the female-lethal gene; and (e) on a large-scale, release ACP males transgenic for a female-lethal trait into orchards with a reduced ACP population.
Related information and references: An alternative reproductive sterility system was developed for the medfly (Ceratitis capitata) based on transgenic embryonic lethality. About 60 transgenic constructions were tested, of which several lines developed larval and pupal lethality. A line that showed complete embryonic lethality nevertheless was highly competitive against wildtype medfly in cage tests (Schetelig et al., 2009).
Suggested support mechanism: This area is NOT recommended for support because there is no indication that psyllids can be reared on a synthetic diet, let alone on a large scale. However, development of an artificial diet for use in large-scale rearing of ACP could justify support. Even if large-scale rearing were to be achieved, the future possible research projects indicated above present tremendous technological challenges.
Time to outcome: Long-term.
Other sections of this report containing related information: Chapter 3, p. 71 (sterile insect technique).